The objective of this proposed project is to synthesize and clone human insulin gene. The project will involve the follwing steps: 1. To synthesize two DNA duplexes, one corresponding to the coding region of human insulin A chain, and the other corresponding to the B chain. 2. To synthesize two oligodeoxynucleotide duplexes, one containing a start and the other a stop signal for protein synthesis. 3. To join by ligation the DNA coding for the insulin A chain to the start and stop signals. Similar joining will be carried out for the B chain. 4. The product from steps 1-3 will be joined to a cloning vehicle, adjacent to a strong promoter. 5. To clone the DNA ligation products from step 4 in E. coli and to identify the clones which contain the insulin gene by hybridization with 32P-labeled single-stranded DNA from step 1. 6. To identify insulin mRNA synthesized in E. coli that carries the putative cloned insulin gene. 7. To isolate, purify and characterize the insulin A-chain and B-chain protein products synthesized by E. coli carrying the cloned insulin gene. To produce biologically active human insulin by recombining the A chain and B chain isolated from step 7.